Team:Kyoto/Notebook

From 2010.igem.org

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Revision as of 04:48, 8 September 2010

LastModified: 2010-9-8

Index

Notebook

Tuesday, July 20

By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

1. Solubilization of antibiotics.

for Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).
for Kanamycin(kan): add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml).
Dispense 1.1ml of the solution into 1.5ml tubes.
Store in the freezer (-20℃).

2. Making plates for LB (Amp+) and LB (Kan+).

3. Transformation of iGEM Parts.

Name	Well	Sample (µl)	Competent Cells (µl)	Total (µl)	Plate	Incubation	Result
<partinfo>J23100</partinfo>	1-18-C	1	20	21	LB (Amp+)	At 37℃ 7/20 20:50 - 7/21 17:00	○
<partinfo>J23105</partinfo>	1-18-M	1	20	21			○
<partinfo>J23116</partinfo>	1-20-M	1	20	21			○
<partinfo>R0011</partinfo>	1-6-G	1	20	21			○
<partinfo>E0840</partinfo>	1-12-O	1	20	21			○
<partinfo>J06702</partinfo>	2-8-E	1	20	21			○
<partinfo>pSB4K5</partinfo>	1-5-G	1	20	21			×
<partinfo>B0015</partinfo>	1-23-L	1	20	21	LB (Kanamycin+)		×

"1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
Discussion
A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".

Wednesday, July 21

By: Wataru, Ken, Makoto, Takuya Yamamoto

1. Culture plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.

2. Make a master plate of the above plates.

3. Retry Transformation of iGEM Parts.

Name	Well	Sample (µl)	Competent Cells (µl)	Total (µl)	Plate	Incubation	Result
<partinfo>pSB4K5</partinfo>	1-5-G	1	20	21	LB (Kanamycin+)	At 37℃ 7/21 20:50 - 7/22 16:30	○
<partinfo>B0015</partinfo>	1-23-L	1	20	21	LB (Kanamycin+)	At 37℃ 7/21 20:50 - 7/22 16:30	○

4. PCR for S-R-Rz/Rz1 and S

Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.

No.	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	TemplateDNA (5ng/µl)	Primer S-R-Rz/Rz1 Forward (10µM)	Primer S-R-Rz/Rz1 Reverse (10µM)	Primer S Reverse (10µM)	KOD Plus ver.2	Total
1	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
2	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
3	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
4	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
5	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
6	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
7	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
8	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl

Forward Primer of S-R-Rz/Rz1 and S is common.
PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

Thursday, July 22

By: Wataru

1. Electrophoresis of the PCR products for 40min.

File:KyotoEXP100722-1.png

Discussion
Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.

2. Miniprep of iGEM Parts.

Name	Concentration(ng/µl)
<partinfo>J23100</partinfo>	18.5
<partinfo>J23105</partinfo>	12.5
<partinfo>J23116</partinfo>	14.6
<partinfo>R0011</partinfo>	8.6
<partinfo>E0840</partinfo>	12.1
<partinfo>J06702</partinfo>	14.7

Discussion
The concentration of all samples was very week. Probably our shaking incubation was week.

3. Culture plates and make master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 7/22 17:00 to 7/23 10:00.

Friday, July 23

By: Wataru, Tomo, Makoto

1. Miniprep of iGEM Parts.

Name	Concentration(ng/µl)
<partinfo>pSB4K5</partinfo>	79.2
<partinfo>B0015</partinfo>	-

Discussion
We lost <partinfo>B0015</partinfo> by our mistake.
The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.

2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.

Sample	Concentration (ng/µl)	New Name
1	18.6	-
3	77.6	S₁
5	33.6	-
7	65.4	S₂

Discussion
The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.

3. Retry of PCR of S-R-Rz/Rz1.

Sample	Water	25mmol/l MgSO4	2mmol/l dNTPs	10×Buffer for KOD plus ver.2	Template DNA (5ng/µl)	Primer S-R-Rz/Rz1 Forward (10µmol/l)	Primer S-R-Rz/Rz1 Reverse (10µmol/l)	KOD plus ver.2	Total
1	28µl	3	5	5	5	1.5	1.5	1	50
2	28	3	5	5	5	1.5	1.5	1	50
3	26.5	4.5	5	5	5	1.5	1.5	1	50
4	26.5	4.5	5	5	5	1.5	1.5	1	50
5	25	6	5	5	5	1.5	1.5	1	50
6	25	6	5	5	5	1.5	1.5	1	50

PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.

Sample	10xBuffer	BSA	Enzyme	MilliQ	Total	Incubation
1	5µl	1	EcoRI 0.1	3.6	10	At 37℃ 7/23 18:00 - 7/23 18:30
2	5	1	XbaI 0.1	3.6	10
3	5	1	SpeI 0.1	3.6	10
4	5	1	PstI 0.1	3.6	10
5	5	1	-	3.7	10

5. Electrophoresis of above sample for 35min.

Discussion
Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

6. To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.

Sample	10×Buffer	Enzyme 1	Enzyme 2	MilliQ	Total	Incubation
S₁	11µl	5	EcoRI 0.2	SpeI 0.2	33.6	50	At 37℃ for 2h
S₂	11	5	EcoRI 0.2	SpeI 0.2	33.6	50
<partinfo>E0840</partinfo>(GFP)	45	5	EcoRI 0.2	XbaI 0.2	0	50

After PCR purification, evaporated them and diluted 3ul.

7. Ligated over night.

Sample	Vector	Insert	Ligation High	Total
S-GFP₁	<partinfo>E0840</partinfo> 0.5µl	S₁ 0.5	1	2
S-GFP₂	<partinfo>E0840</partinfo> 0.5	S₂ 0.5	1	2

Monday, July 26

By: Wataru, Tomonori, Makoto

1. Electrophoresis of PCR products

Discussion
At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.

2. PCR purification

Sample	Concentration (ng/µl)	New Name
4	51.6
5	59.3
6	59.6

3. Transformation of iGEM Parts

Well	Sample (µl)	Competent Cell (µl)	Total (µl)	Plate	Incubation	Result
1-12-M	1	20	21	LB (Amplicillin+)	At 37℃ 7/26 - 7/27	×
2-17-F	1	20	21	LB (Kanamycin+)		×
1-5-E	1	20	21	LB (Kanamycin+)		×

@@ Line 163: / Line 163: @@
 |5||5||1||-||3.7||10
 |}
-|-
 ====5. Electrophoresis of above sample for 35min.====
 [[Image:KyotoExp100723-1.png|right]]

Team:Kyoto/Notebook

From 2010.igem.org

Revision as of 04:48, 8 September 2010

Contents

Index

Notebook

Tuesday, July 20

1. Solubilization of antibiotics.

2. Making plates for LB (Amp+) and LB (Kan+).

3. Transformation of iGEM Parts.

Wednesday, July 21

1. Culture plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.

2. Make a master plate of the above plates.

3. Retry Transformation of iGEM Parts.

4. PCR for S-R-Rz/Rz1 and S

Thursday, July 22

1. Electrophoresis of the PCR products for 40min.

2. Miniprep of iGEM Parts.

3. Culture plates and make master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 7/22 17:00 to 7/23 10:00.

Friday, July 23

1. Miniprep of iGEM Parts.

2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.

3. Retry of PCR of S-R-Rz/Rz1.

4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.

5. Electrophoresis of above sample for 35min.

6. To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.

7. Ligated over night.

Monday, July 26

1. Electrophoresis of PCR products

2. PCR purification

3. Transformation of iGEM Parts

4. Culture of 1-6-G, 1-12-O, and 1-23-L=